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. 2014 Nov 25;5(6):e02141-14. doi: 10.1128/mBio.02141-14

FIG 3 .

FIG 3 

Inhibition of glutamine synthesis prevents E. chaffeensis growth. (A) MSX inhibits E. chaffeensis growth in THP-1 cells in a dose-dependent manner. DNA samples prepared from synchronously cultured E. chaffeensis in THP-1 cells treated at 0 h p.i. with 20, 100, or 200 µM MSX for 72 h were subjected to qPCR analysis. The values reflect bacterial 16S rRNA gene normalized against human GAPDH DNA, relative to the amount determined for the sham-treated control (CTL). Data indicate the means ± standard deviations from three independent experiments performed in triplicate. (B) Inhibitory effect of MSX is reversible. MSX (200 µM) was added to E. chaffeensis-infected THP-1 cells at 0 h p.i. and removed after 24 or 48 h p.i. DNA samples were subjected to qPCR analysis. The values reflect bacterial 16S rRNA gene normalized against human GAPDH DNA, relative to the amount determined at 0 h p.i. Symbols: ×, MSX was not removed; □, MSX was removed at 24 h p.i.; ▲, MSX was removed at 48 h p.i. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *, significantly different (P < 0.05; analysis of variance) compared with the value of the sample with MSX not removed. (C) Glutamine reverses the inhibitory effect of MSX. DNA samples prepared from synchronously cultured E. chaffeensis in THP-1 cells treated at 0 h p.i. with 200 µM MSX and 10, 20, or 40 mM glutamine (left) or glutamate (right) for 72 h were subjected to qPCR analysis. The values reflect bacterial 16S rRNA gene normalized against human GAPDH DNA, relative to the amount determined for the sham-treated control (CTL). Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *, significantly different (P < 0.05; analysis of variance) compared with the value of the control. (D) MSX has no effect on C. trachomatis growth in L929 cells. C. trachomatis- or E. chaffeensis-infected L929 cells were treated with 200 µM MSX for 72 h. Bacterial counts were scored from 100 cells and are relative to the amount determined for the sham-treated control (CTL). Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *, significantly different (P < 0.05; Student’s t test) compared with the value of the control.